During FY14 we accomplished the following: 1. We carried out the first live cell visualization of IgH locus movement in primary pro-B cells. For this bone marrow pro-B cells from the previously described Site 4 mouse strain were expanded on OP9 stromal cells and infected with retroviruses that express the Tet-repressor DNA binding domain fused to EGFP or mCHERRY fluorescent proteins. Live cell visualization was carried out at NCI in collaboration with Drs. Tatiana Karpova and Tom Misteli. We also generated mouse embryo fibroblasts from Site 4 mice for analysis of IgH locus movements in a non-lymphoid nuclear environment. 2. We studied the molecular mechanism of recombination dys-regulation in cells with mutated CTCF binding sites in the IGCR1 regulatory element of the IgH locus. We found that the intronic enhancer, E, loops to an alternate site close to the 3VH genes on IGCR1 mutated pro-B cells. This results in increased epigenetic changes and transcription close to these VH gene segments and brings these segments close to the RAG1/2-rich recombination center. We propose that this altered proximity may underlie the breakdown of the timing of recombination observed in this strain. Ongoing studies are aimed at testing the role of chromatin structural proteins CTCF and YY1 in this process. These studies are an ongoing collaboration with Dr. Fred Alt (Childrens Hospital, Boston, MA). 3. We completed the first phase of 3D-FISH studies to validate and extend 5C analysis of the VH region of the IgH locus. Three prominent structural sites were identified and interaction between them confirmed by fluorescence microscopy in pro-B cells. FISH studies were extended to E-deficient and Pax5-deficient pro-B cells and to non-lymphoid hematopoietic cells. Interactions between these sites were found to be B lineage-specific, and Pax5-dependent, but E-independent. Mutational studies of these interaction sites are planned for the future. These studies were carried out in collaboration with Dr. Amy Kenter (University of Illinois Medical Center). 4. We identified a novel DNAase I hypersensitive located towards the 3 end of J558 VH gene family. Protein binding and mutational studies have been initiated.